Exendin-4 is a 39 amino acid peptide which is produced by the salivary glands of the Gila monster (Heloderma suspectum) (Eng J. et al., J. Biol. Chem., 267:7402-05, 1992). Exendin-4 is an activator of the glucagon-like peptide-1 (GLP-1) receptor, whereas it shows only very low activation of the GIP receptor and does not activate the glucagon receptor (see Table 1).
TABLE 1Potencies of exendin-4 at human GLP-1, GIP and Glucagonreceptors (indicated in pM) at increasing concentrations and measuring the formed cAMP as described in Methods.SEQ IDEC50 hGLP-1 REC50 hGIP REC50 hGlucagonNO:peptide[pM][pM]R [pM]1exendin-4 0.412500.0>10000000
Exendin-4 shares many of the glucoregulatory actions observed with GLP-1. Clinical and non-clinical studies have shown that exendin-4 has several beneficial antidiabetic properties including a glucose dependent enhancement in insulin synthesis and secretion, glucose dependent suppression of glucagon secretion, slowing down gastric emptying, reduction of food intake and body weight, and an increase in beta-cell mass and markers of beta cell function (Gentilella R et al., Diabetes Obes Metab., 11:544-56, 2009; Norris S L et al., Diabet Med., 26:837-46, 2009; Bunck M C et al., Diabetes Care., 34:2041-7, 2011).
These effects are beneficial not only for diabetics but also for patients suffering from obesity. Patients with obesity have a higher risk of getting diabetes, hypertension, hyperlipidemia, cardiovascular and musculoskeletal diseases.
Relative to GLP-1 and GIP, exendin-4 is more resistant to cleavage by dipeptidyl peptidase-4 (DPP4) resulting in a longer half-life and duration of action in vivo (Eng J., Diabetes, 45 (Suppl 2):152A (abstract 554), 1996; Deacon C F, Horm Metab Res, 36: 761-5, 2004).
Exendin-4 was also shown to be much more stable towards degradation by neutral endopeptidase (NEP), when compared to GLP-1, glucagon or oxyntomodulin (Druce M R et al., Endocrinology, 150(4), 1712-1721, 2009).
Nevertheless, exendin-4 is chemically labile due to methionine oxdiation in position 14 (Hargrove D M et al., Regul. Pept., 141: 113-9, 2007) as well as deamidation and isomerization of asparagine in position 28 (WO 2004/035623).
The amino acid sequence of exendin-4 is shown as SEQ ID NO: 1:
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2
The amino acid sequence of GLP-1(7-36)-amide is shown as SEQ ID NO: 2:
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
Liraglutide is a marketed chemically modified GLP-1 analogue in which, among other modifications, a fatty acid is linked to a lysine in position 20 leading to a prolonged duration of action (Drucker D J et al, Nature Drug Disc. Rev. 9, 267-268, 2010; Buse, J B et al., Lancet, 374:39-47, 2009).
The amino acid sequence of Liraglutide is shown as SEQ ID NO: 3:
HAEGTFTSDVSSYLEGQAAK((S)-4-Carboxy-4-hexadecanoyl- amino-butyryl-)EFIAWLVRGRG-OH
GIP (glucose-dependent insulinotropic polypeptide) is a 42 amino acid peptide that is released from intestinal K-cells following food intake. GIP and GLP-1 are the two gut enteroendocrine cell-derived hormones accounting for the incretin effect, which accounts for over 70% of the insulin response to an oral glucose challenge (Baggio L L, Drucker D J. Biology of incretins: GLP-1 and GIP. Gastroenterology 2007; 132: 2131-2157).
GIP's amino acid sequence is shown as SEQ ID NO: 4:
YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-OH
Glucagon is a 29-amino acid peptide which is released into the bloodstream when circulating glucose is low. Glucagon's amino acid sequence is shown in SEQ ID NO: 5:
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH
During hypoglycemia, when blood glucose levels drop below normal, glucagon signals the liver to break down glycogen and release glucose, causing an increase of blood glucose levels to reach a normal level. Hypoglycemia is a common side effect of insulin treated patients with hyperglycemia (elevated blood glucose levels) due to diabetes. Thus, glucagon's most predominant role in glucose regulation is to counteract insulin action and maintain blood glucose levels.
Hoist (Hoist, J. J. Physiol. Rev. 2007, 87, 1409) and Meier (Meier, J. J. Nat. Rev. Endocrinol. 2012, 8, 728) describe that GLP-1 receptor agonists, such as GLP-1, liraglutide and exendin-4, improve glycemic control in patients with T2DM by reducing fasting and postprandial glucose (FPG and PPG). Peptides which bind and activate the GLP-1 receptor are described in patent applications WO 98/08871 A1, WO2008/081418 A1 and WO2008/023050 A1, the contents of which are herein incorporated by reference.
It has been described that dual activation of the GLP-1 and GIP receptors, e.g. by combining the actions of GLP-1 and GIP in one preparation, leads to a therapeutic principle with significantly better reduction of blood glucose levels, increased insulin secretion and reduced body weight in mice with T2DM and obesity compared to the marketed GLP-1 agonist Liraglutide (e.g. V A Gault et al., Clin Sci (Lond), 121, 107-117, 2011). Native GLP-1 and GIP were proven in humans following co-infusion to interact in an additive manner with a significantly increased insulinotropic effect compared to GLP-1 alone (M A Nauck et al., J. Clin. Endocrinol. Metab., 76, 912-917, 1993).
Designing hybrid molecules which combine agonism on the GLP-1 receptor, the GIP receptor and the glucagon receptor offers the therapeutic potential to achieve significantly better reduction of blood glucose levels, increased insulin secretion and an even more pronounced significant effect on body weight reduction compared to the marketed GLP-1 agonist Liraglutide (e.g. V A Gault et al., Clin Sci (Lond), 121, 107-117, 2011).
Compounds of this invention are exendin-4 derivatives, which show agonistic activity at the GLP-1 and the GIP receptor and optionally the glucagon receptor and which have—among others—preferably the following modifications: Tyr at position 1 and Ile at position 12.
Surprisingly, it was found that the modification of the selective GLP-1R agonist Exendin-4 by Tyr in position 1 and Ile in position 12 results in a peptide with high dual activity at the GLP-1 and GIP receptors. This observation is surprising, since the same modification in other GLP-1 agonists, such as GLP-1 itself, does not result in high activity at the GIP receptor, as shown in Table 2.
TABLE 2Potencies of exendin-4 and GLP-1 peptide analogues at GLP-1 andGIP receptors (indicated in pM) at increasing concentrations and measuring the formed cAMP as described in Methods.SEQ EC50 EC50 IDhGIP RhGLP-1 RNO:peptide[pM][pM]6Tyr(1)Ile(12)-exendin-493.91.37Tyr(1)Ile(12)-GLP13660.05.0
Peptides which bind and activate both the GIP and the GLP-1 receptor and optionally the glucagon receptor, and improve glycaemic control, suppress body weight gain and reduce food intake are described in patent applications WO 2011/119657 A1, WO 2012/138941 A1, WO 2010/011439 A2, WO 2010/148089 A1, WO 2011/094337 A1, WO 2012/088116 A2, the contents of which are herein incorporated by reference. These applications disclose that mixed agonists of the GLP-1 receptor, the GIP receptor and optionally the glucagon receptor can be designed as analogues of the native GIP or glucagon sequences.
Compounds of this invention are exendin-4 peptide analogues comprising leucine in position 10 and glutamine in position 13. Krstenansky et al. (Biochemistry, 25, 3833-3839, 1986) show the importance of residues 10 to 13 of glucagon for its receptor interactions and activation of adenylate cyclase. In the exendin-4 peptide analogues of this invention, several of the underlying residues are different from said of glucagon. In particular, residues Tyr10 and Tyr13, are replaced by leucine in position 10 and glutamine, a non-aromatic polar amino acid, in position 13. This replacement, especially in combination with isoleucine in position 23 and glutamate in position 24 leads to exendin-4 derivatives with potentially improved biophysical properties as solubility or aggregation behavior in solution. The non-conservative replacement of an aromatic amino acid with a polar amino acid in position 13 of an exendin-4 analogue surprisingly leads to peptides with high activity on the GIP receptor and optionally on the glucagon receptor.
Furthermore, compounds of this invention are exendin-4 derivatives with fatty acid acylated residues in position 14. This fatty acid functionalization in position 14 results in an improved pharmacokinetic profile. Surprisingly, the fatty acid functionalization in position 14 also leads to peptides with a significantly higher GIPR activity, for example those shown in Example 5, Table 8.
Compounds of this invention are exendin-4 peptide analogues which contain alpha,alpha-dialkylated amino acids with a basic side-chain in position 20. Surprisingly, modification of the exendin-4 sequence with one of these amino acids leads to compounds with an improved biophysical profile, as solubility (in particular at low pH, especially at pH 4.5) or aggregation behaviour in solution, when the unnatural amino acid is incorporated at position 20. The resulting exendin-4 analogues thereby maintain their high activity at the GLP-1 receptor, the GIP receptor and optionally the glucagon receptor. The incorporation of these unnatural amino acids also increases enzymatic stability of the peptides, potentially resulting in improved pharmacokinetic properties.